Plant extracts and uses thereof in filter systems

ABSTRACT

The present invention relates to compositions and methods for reducing the concentration of noxious substances in gases and smoke. Filters comprising plant extracts, such as parenchyma tissue extract and mesophyll cell extract, are also provided, as well as methods for making same.

CROSS-RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application 60/785,974 filed Mar. 27, 2006, the entire content of this application being hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the reduction of toxicity and particularly to cellular toxicity of fumes. The present invention also relates to compositions and devices for reducing the concentration of one or more noxious substances in fumes, particularly in smoke, and more particularly in cigarette smoke.

BACKGROUND OF THE INVENTION

Parenchyma cells serve various structural and biochemical functions in plants. They are the bulk of ground tissue and vascular tissue in plants. Palisade parenchyma cells are elongated cells located in leaves just below the epidermal tissue. Ray parenchyma cells are located in wood rays, the structures transporting materials laterally within a woody stem. Parenchyma cells also occur within the xylem and phloem of vascular bundles. The largest parenchyma cells are located in the pith region, being often larger than the vascular bundles, as in corn stems. These cells are the biochemistry machines of the plant, regulating light penetration, gas exchange, and anti-herbivory physiology for example. Other cells, such as mesophyll cells, are specialized in photosynthesis or phloem loading. Spongy mesophyll cells are located below one or two layers of palisade parenchyma cells.

Chloroplasts are membranous organelles found in plant cells that serve as site of photosynthesis. Typically, chloroplasts comprise three types of membranes, which are: (i) a smooth outer membrane, which is freely permeable to molecules; (ii) a smooth inner membrane, which contains many transport proteins such as integral membrane proteins regulating the exchange of small molecules like sugars and proteins between the cytoplasm and the chloroplast; and (iii) a system of thylakoid membranes which contains the chlorophyll.

Filters for filtering toxic combustion fumes have not always been a priority in industrial, commercial or house environment, and inhalation of toxic or cytotoxic fumes, including from smoking, can cause serious health problems that can lead to death. About 87% of lung cancer deaths are attributed to air pollutant particles and smoke, or smoking. For example, cigarette smoking and second-hand smoke account for at least 30% of all cancer deaths. It is a major cause of cancers of the lung, larynx, oral cavity, pharynx (throat), and esophagus, and is a contributing cause in the development of cancers of the bladder, pancreas, liver, uterine, cervix, kidney, stomach, colon and rectum, as well as in some leukemia. Lung cancer is the leading cause of cancer death among both men and women, and is one of the most difficult cancers to treat. In addition, this type of cancer is very hard to detect when it is in its earliest, most treatable stage. Smoking is also a major cause of heart disease, bronchitis, emphysema and stroke, and it contributes to the severity of pneumonia. Tobacco also has a damaging effect on women's reproductive health and is associated with increased risk of miscarriage, early delivery, stillbirth, infant death, in addition of being a cause of low birth weight in infants. Furthermore, cigarette smoke causes harmful effects on people indirectly exposed to the smoke (second-hand smoke). The diseases occurring most often from second-hand smoke are chronic bronchitis, emphysema, heart attacks, strokes, and cancer.

Cigarette, cigar and pipe tobacco consist of dried tobacco leaves supplemented with additional ingredients modifying tobacco flavor or having other properties. More than 4,000 individual substances and compounds have been identified in tobacco and tobacco smoke. It is known that tobacco-specific nitrosamines and tar are particularly carcinogenic, while carbon monoxide decreases the amount of oxygen in the blood. A decreased blood oxygen often leads to an increase of the heartbeat and blood pressure, two conditions that can translate into shortness of breath. In addition to being a carcinogen, tar condenses itself as a brown sticky substance that can accumulate into the lungs in such a way that it eventually clogs up the cilia protecting and cleaning the lungs, accompanied in this process by many other noxious substances present in the resulting smoke being inhaled.

A way to reduce the toxic effect of tobacco smoke is the addition of filters to cigarettes. Ninety-five percent of cigarette filters are made of cellulose acetate, while the other five percent are made of paper and rayon. Cellulose acetate tow fibers used in cigarette filters are thinner than sewing thread, usually white, and packed tightly together to form a physical filter that resemble cotton.

A solution for reducing the toxic effect of tobacco smoke has been proposed by Hersh et al (U.S. Pat. No. 6,470,894), which describes a composition comprising glutathione with green tea and/or grape seed extract. This composition can be placed within a cigarette filter, cigarette wrapping paper, or directly into the tobacco for reducing the concentration of free radicals in the tobacco smoke. Stavridis et al. (U.S. Pat. No. 5,909,736) describes a method of withholding noxious substances from tobacco smoke by adding biological sources of metal ions, such as hemoglobin or lysates of erythrocytes, to a cigarette filter. Rivers (U.S. Pat. No. 5,387,285) describes an apparatus for injecting a fluid into a filter such as a cigarette filter. Agarie et al. (U.S. Pat. No. 5,083,578) describes a filter containing seaweed to unspecifically remove toxic material from tobacco smoke. Takanashi (U.S. Pat. No. 4,756,319) describes a process for producing granular algal adsorbents that can be used for removing toxic and odiferous materials in smoke and in air. Pryor (U.S. Pat. No. 4,525,385) describes a process for applying an additive to a filter tow.

Noxious substances can also be found in the air or in fumes absorbable by living organisms, such as in gases released in the air by industrial activity of mining or incineration industries for example. To reduce the exposure to such noxious substances, and to prevent their pulmonary absorption in workers of such industries, gas masks including filters are often used. In fact, in some professions, national laws increasingly require that gas masks be worn by workers who, in their daily work, are exposed to potentially harmful chemicals, as for example painters and fitters in the building profession. Gas masks currently use mainly means of adsorbing noxious substances onto a layer of activated charcoal. However, the efficiency of such filters decreases over time with the saturation of the activated carbon layer.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the lung that is characterized by progressive and irreversible airflow limitation. Cigarette smoking has been identified as the major risk factor for the apparition and development of COPD. The inflammatory cell profile in COPD includes macrophages, neutrophils, and T lymphocytes.

Macrophages are blood cells that are present in several animal tissues. They accomplish three principal functions in the tissues, namely the synthesis of cytokines, the regulation of cellular cooperation, and the phagocytosis of foreign molecules. Following pulmonary inflammation and parenchymal damage, for example during COPD, there is an increased concentration of lung alveolar macrophages (AM) that release various cytokines and proteases when compared to non-COPD lungs. AM also contribute to airway inflammation in smokers and COPD patients by secreting neutrophil and macrophage chemotactic factors such as interleukins and chemokines; by secreting cell survival factors suck as granulocyte macrophage-colony stimulating factor (GM-CSF); and by generating reactive oxygen species. Reducing the concentration of noxious substances in lungs would lead to a reduced migration of macrophages towards the alveolus, and thus reduce pulmonary inflammation.

Although different approaches have been tested and commercialized in the art, there is still a need for an efficient composition and method for removing or reducing noxious substances from fumes, gases and smoke, in order to provide an environment less damaging for health, particularly health of lungs exposed to such noxious substances.

SUMMARY OF THE INVENTION

The problem to which the present invention provides a solution is the reduction of the concentration of noxious substances in fumes by filtering through a filter containing a biological material.

One aspect of the present invention is to provide a plant extract for reducing the concentration of noxious substances in a fume, or for reducing the toxicity or cytotoxicity of a fume, the plant extract comprising at least one of a parenchyma tissue extract (PTE) and a mesophyll cell extract (MCE).

In accordance with the present invention, the term “plant extract” as used herein is intended to mean any crude, processed or refined plant extract. This term also encompass any plant portion or derivative from which can be obtained a plant extract, a PTE, a MCE or a mixture thereof.

In further accordance with the present invention, the term “fume” (or “fumes”) as used herein is intended to represent any combustion, environmental, automotive, domestic, commercial or industrial gas, as well as smoke, including cigarette smoke. Also, depending on the environment, a gas can be found under the form of a vapor, which is therefore encompassed herein. Non-limitative examples of fumes includes fumes produced by industrial activity, by burning apparatuses, or by animals or plants, such as in an animal or plant production plant. This also includes environmental fumes produced in a commercial place, such as in a restaurant, a commercial center, a public kitchen, a laboratory, or a workshop. This is also intended to represent domestic fumes, such as those produced in a kitchen, as well as fumes produced by automotive means, such as a car, a truck, a train, or a boat. The term “fume” as used herein is also meant to encompass smoke, such as tobacco smoke and cigarette smoke. The terms “toxic”, “cytotoxic”, “toxicity” and “cytotoxicity” are used in the present application to reflect the capacity of a fume, or of a compound or substance from the fume, to have a poisonous, deleterious, noxious or lethal effect on living cells or organisms. Toxicity and cytotoxicity can either come from direct exposure to the fume, or from indirect exposure, such as second-hand smoke or environmental gases.

In still further accordance with the present invention, the expression “parenchyma tissue extract” (PTE) as used herein is intended to mean a plant extract that can comprises, without being restricted to, the cortex and pith of stems, the cortex of roots, the mesophyll, the pulp of fruits, the endosperm of seeds, the chlorenchyma and palisade tissue of leaves, thylakoid systems, including chloroplasts. PTE also comprises vascular cells from the xylem and the phloem.

The expression “mesophyll cell extract” (MCE) as used herein is intended to represent the filtrate obtained from the filtration of a PTE that has been further processed so as to remove bundle sheath cells, vascular cells and cell walls.

In accordance with the present invention, the plant extract comprising at least one of a PTE and a MCE has a total chlorophyll concentration ranging from 0.03 to 1100 ng/g, preferably from 0.03 to 500 ng/g, more preferably from 0.05 to 200 ng/g, and even more preferably from 0.05 to 50 ng/g of dried plant extract. The expression “total chlorophyll concentration” as used herein is intended to represent the concentration of chlorophyll-a and chlorophyll-b, calculated according to the formula:

$\frac{\begin{matrix} {\left( {7.15 \times {Absorbance}\mspace{14mu} {at}\mspace{14mu} 663.2\mspace{14mu} {nm}} \right) +} \\ \left( {18.71 \times {Absorbance}\mspace{14mu} {at}\mspace{14mu} 646.8\mspace{14mu} {nm}} \right) \end{matrix}}{1000}$

This formula expresses the total chlorophyll concentration in mg/ml.

In yet further accordance with the present invention, the expression “noxious substances” as used herein is intended to represent any substance or compound that can be identified from fumes by any analytical means, and that can have a poisonous, deleterious, undesirable, noxious or lethal effect on living cells or organisms, alone or in combination with another substance or compound, either from direct or indirect exposure.

Another aspect of the present invention is a method for reducing the concentration of noxious substances in a fume, or for reducing the toxicity or cytotoxicity of a fume, by contacting the fume with a plant extract comprising at least one of a PTE and a MCE.

Another aspect of the present invention is to provide the use of a plant extract for reducing the concentration of noxious substances in a fume, or for reducing the toxicity or cytotoxicity of a fume, the plant extract comprising at least one of a PTE and a MCE.

Still another aspect of the present invention is to provide a composition for reducing the concentration of noxious substances in a fume, or for reducing the toxicity or cytotoxicity of a fume, the composition comprising a plant extract comprising at least one of a PTE and a MCE; and a carrier material.

In accordance with the present invention, the term “carrier material” as used herein is intended to represent any type of material compatible with the intended use, on which a molecule, a compound, a substance (such as a plant extract) or a composition can be supported, either by physical interaction or by chemical interaction with the carrier material. As an example of the compatibility of a carrier material to an intended use, a carrier material to be used as a filter mean with a plant extract or a composition as defined herein has to allow fumes to pass through the filter such that the fumes come in contact with the plant extract or the composition. As an example of the interaction of the carrier material with a plant extract or a composition, the interaction has to not prevent the plant extract or composition to exert its desirable effect, such as reducing the concentration of noxious substances in a fume, or for reducing the toxicity or cytotoxicity of the fume.

Another aspect of the present invention is a method for reducing the concentration of noxious substances in a fume, or for reducing the toxicity or cytotoxicity of a fume, by contacting the fume with a composition comprising a plant extract comprising at least one of a PTE and a MCE; and a carrier material.

Another aspect of the present invention is to provide the use of a composition for reducing the concentration of noxious substances in a fume, or for reducing the toxicity or cytotoxicity of a fume, the composition comprising a plant extract comprising at least one of a PTE and a MCE; and a carrier material.

Yet another aspect of the present invention is to provide a filter for reducing the concentration of noxious substances in a fume, or for reducing the toxicity or cytotoxicity of a fume, the filter comprising a carrier material and a plant extract comprising at least one of a PTE and a MCE, with the plant extract being disposed within the carrier material in a way to allow contact of the fume with the plant extract when the fume is circulating through the filter.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration preferred embodiments in which:

FIG. 1. illustrates the reversal of the inhibition of inflammatory mediators caused by tobacco smoke by exogenous addition of plant extracts. Alveolar macrophages (AM) were treated with cigarette smoke extract alone (CSE), CSE+PTE and CSE+MCE for 20 h and the mediators tumor-necrosis factor-α (TNFα), interleukin 10 (IL-10) and monocyte chemotactic protein 1 (MCP-1) were measured in cell-free supernatants. CSE significantly (*p<0.02) inhibited the release of (a) TNFα, (b) IL-10, and (c) MCP-1 by AM. Exposure of AM to plant extract (PTE or MCE) with CSE eliminated this inhibition. Both concentrations of PTE and MCE (0.0025% and 0.025%) significantly (#p<0.01) stimulated the release of TNFα, whereas only high concentrations of MCE (0.025%) significantly (#p<0.01) increased the release of IL-10 and MCP-1. Mean±s.e.m. of 6 experiments;

FIG. 2. illustrates the restoration of AM cytotoxicity towards tumoral cells by MCE. AM were treated with CSE, CSE+PTE and CSE+MCE for 20 h and the cytotoxicity of AM towards against WEHI-164 tumoral cells was investigated (ratio AM:WEHI-164 of 50:1). CSE significantly (*p<0.05) inhibited AM cytotoxicity under those conditions. The presence of PTE did not modulate CSE inhibition, whereas MCE restored AM cytotoxicity towards tumoral cells. Mean±s.e.m. of 5 experiments;

FIG. 3. illustrates the modulation of AM mediator production by CSE after filtration by a plant extract-containing cigarette filter. CSE with PBS buffer (“buffer”), CSE with 0.025% and 0.25% PTE (“PTE”) and CSE with 0.025% and 0.25% MCE (“MCE”) were applied to AM for 20 h. Following that, mediators (a) TNFα, (b) IL-10 and (c) MCP-1 were measured in cell-free supernatants. CSE filtrated through the buffer-treated filter significantly (*p<0.02) inhibited the release of TNFα, IL-10, and MCP-1. CSE filtrated through the MCE-treated cigarette filter (0.25%) abrogated the inhibitory effects of CSE on mediator production. Mean±s.e.m. of 6 experiments; and

FIG. 4. illustrates the modulation of AM cytotoxicity towards tumoral cells by a plant extract-containing cigarette filter. CSE with PBS buffer (“buffer”), CSE with 0.025% and 0.25% PTE (“PTE”) and CSE with 0.025% and 0.25% MCE (“MCE”) were applied to AM for 20 h. Following that, the cytotoxicity of AM towards against WEHI-164 tumoral cells was investigated (ratio AM:WEHI-164 of 50:1). CSE filtrated through the buffer-treated cigarette filter significantly (*p<0.05) inhibited AM cytotoxicity towards tumoral cells. CSE filtrated through the PTE-treated cigarette filter (0.25%) and the MCE-treated cigarette filter (0.025% and 0.25%) restored AM cytotoxicity towards tumoral cells. Mean±s.e.m. of 6 experiments.

FIG. 5 illustrate the reduction of the amount of the heavy metals: cadmium, lead, nickel, chromium and mercury in the smoke of regular and king size cigarettes. Dark bars represent heavy metals concentration in a control cigarette equipped with a regular filter, while light bars represent heavy metals concentration in a cigarette equipped with a plant extract-containing filter. There is significant reduction in the concentration of heavy metals concentration in smoke from cigarettes equipped with a plant extract-containing filter when compared with the smoke from cigarettes equipped with a regular filter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention, may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Different plant species can be used to prepare plant extracts. For example, spinach leaves are interesting for preparing plant extracts according to the present invention because they possess a high mesophyll/leaf tissue ratio, present high intrinsic activity (photosynthetic), and their agricultural parameters are well known for reproducibility. However, any plant species can be used in the preparation of the plant extracts according to the present invention, including tobacco, algae, bryophyta, and vascular plants.

Plant extracts can be obtained by different methods, such as roughly crunching or homogenizing whole chloroplast-containing organisms, such as whole plants, plant parts or plant tissues such as parenchyma tissue. For example, mechanical homogenization of whole plants, plant parts or plant tissues in a phosphate buffer with sodium chloride results in PTE. The homogenization can be performed on whole plants, plant parts or plant tissues in a relatively dry state, as long as the mesophyll cells are kept intact and functional.

Processing of PTE by filtration, either with centrifugation or application of an equivalent pressure, allows for the removal of bundle sheath cells and various cell walls, and will results in the obtention of MCE. It is of note that different processes are now known in the art for the preparation of PTEs and MCEs.

Without wishing to be bound by theory, PTE and MCE allow for the adsorption and/or absorption of several toxic molecules present in fumes. Adsorption is the process by which a substance or compound is retained on the surface of a component, while absorption is the process by which a substance or compound is taken inside a component (such as a cell). Adsorption and/or absorption is carried out by the membrane constituents presents in PTE and MCE. Those membranes present a double lipid layer linked to macromolecules such as structural or functional proteins. It is of note that PTE and MCE can be combined together to adjust the ratio of membrane constituents present in the plant extract.

In accordance with the present invention, the plant extract comprises intact membrane constituents. It is of note that the expression “membrane constituents” and the expression “cell membranes” are used herein interchangeably. The term “intact” as used herein in relation with membrane constituents reflect the non-disrupted state of the membrane constituents, allowing for the membrane constituents to substantially preserve their native roles and functions. The membrane constituents can include various membranes, such as membranes composed of lipopolysaccharides, usually having a structural role and acting as a physical barrier against outside molecules, while having some degree of permeability to some ions and metabolites. Membrane constituents can also originate from membranes composed of a phospholipids bilayer, usually specialized in transport proteins regulating the transport of selective metabolites. They can also come from the thylakoid system (or thylakoid membranes), which is the site of light absorption and ATP synthesis involved in the conversion of light energy into chemical energy in plants. In order to preserve the integrity of the membrane constituents during homogenization, filtration, isolation or any other procedure, the tonicity of their environment must be strictly controlled. For example, an hypertonic buffer containing 50 mM phosphate pH 7.5 with 10 mM sodium chloride allows for the preservation of the three-dimensional structure of the membrane constituents of chloroplasts. Analysis of the integrity of the membrane constituents can also be performed by techniques known in the art, such as fluorescence, optical spectroscopy, pigment ratio analysis, photosystem II oxygen evolving, photoacoustics, etc.

As non-limitative examples, noxious substances that can be adsorbed or absorbed by the plant extracts of the present invention include peroxidase, superoxide, free radical, aldehyde, polycyclic aromatic hydrocarbon, nitrosamine, secondary amine, ammonia, carbon monoxide, nicotine, nitric oxide, hydrogen cyanide, mercury, tar, nickel, lead, cadmium, chromium, arsenic, selenium, N-nitrosonomicotine, nicotine-derived nitrosamino ketone, N-nitrosoanatabine, N-nitrosoanabasine, 1-aminonapthalene, 2-aminonapthalene, 3-aminobiphenyl, 4-aminobiphenyl, benzo[a]pyrene, formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, methyl ethyl ketone, butyraldehyde, pyridine, quinoline, hydroquinone, resorcinol, catechol, phenol, m+p-cresol, o-cresol, 1,3-butadiene, isoprene, acrylonitrile, benzene, toluene and styrene.

The quantity of plant extract to be incorporated into a filter according to the present invention can be adjusted depending on the needs. For example, but not limited to, a regular cigarette filter, corresponding to a volume of about 0.8 cm³ to 1.2 cm³, may contain between 0.005 mg to 200 mg of plant extract. Concentrations of plant extract in the carrier material, such as in a cigarette filter, are from 0.005 to 200 mg/cm³, preferably from 0.010 to 100 mg/cm³, more preferably from 0.020 to 50 mg/cm³, and even more preferably from 0.025 to 25 mg/cm³, of carrier material. Preferably, when a mixture of PTE and MCE is used, the ratios of PTE:MCE is between 1:1 and 1:9, preferably between 1:1 to 1:5, and more preferably from 1:1 to 1:2. The incorporation of the plant extract into the carrier material, such as a filter, can be carried out by any method known in the art, such as by injection, vaporization, or dipping. Preferentially, the filter must not be saturated in plant extract in order to avoid any alteration or reduction of its efficiency. The plant extract can be under any form for incorporation, such as an homogenate, a filtrate, a retentate, a diluted form, a dried form, or at various humidity concentration. The filter density can also be adjusted to support different concentrations of plant extract, based on, as non-limitative examples, the weight of the filter, the circumference of the filter, and the loss of pressure during use. Alternatively, the concentration of the plant extract can be expressed in equivalent chlorophyll concentration. The total equivalent chlorophyll concentration, for example in a regular cigarette filter, can be from 0.03 to 1100 ng/g, preferably from 0.03 to 500 ng/g, more preferably from 0.05 to 200 ng/g, and even more preferably from 0.05 to 50 ng/g.

The filter in accordance with the present invention can be adapted to fit with a cigarette or with a gas mask, or any other filtration devices or means. The filter should therefore have the capacity to be used in conjunction with, for example, a cigarette or a gas mask so as to effectively reduce the concentration of noxious substances in smoke or fumes originating from the cigarette or the environment and passing through the filter, or for reducing the toxicity or cytotoxicity of smoke or fumes originating from the cigarette or the environment and passing through the filter. For gas mask and other filtrations devices and means, the filter can be secured into a cartridge, or into any other filter carrier body through which the filter is incorporated, as known in the art.

In addition, it will be understood that the filter according to the invention can also be advantageously used as an industrial air filter (domestic and building) or as an automotive car filter (such as exhaust gas purification filter filtering the gas discharged from an internal combustion engine before its release in the environment). Further, the filter can also be used to filter vehicle fumes, or for filtering domestic or industrial smoke or fumes. When a portion of the burned fumes penetrates the vehicle cabin or the operating area, it exposes the driver or operator to inhale the gases containing noxious substances. Hence, a filter according to the present invention can be used for purifying air by reducing the concentration of noxious substances that can be present in the air.

In accordance with the present invention, the carrier material can comprises a porous substrate. The porous substrate can be any non-toxic material suitable for use in filters for smoking devices, or other filtration devices, and is also suitable for incorporation of other substances according to the various embodiments of the present invention. Such porous substrates include, for example, cellulose fibre such as cellulose acetate, cotton, wood pulp, paper, polyesters, polyolefins, ion exchange materials and other materials, as will be understood by those with skill in the art with reference to the disclosure herein.

In accordance with the present invention, the composition can further comprise at least one of an activated charcoal (or activated carbon) or an ion exchange resin, or any other material that can improve for the filtration.

It will be understood by the skilled artisan that the carrier material for the plant extract for environmental applications can be different than the carrier material described above. For example, different plastics, papers, fibers, or any other material known in the art of gas and air purification can be used with the plant extract fixed thereon, in accordance with the present invention, before being placed in any environment where toxic or cytotoxic substances have to be removed from the environment.

All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby incorporated herein by reference.

The present invention will be more readily understood by referring to the following examples, which are given to illustrate the invention rather than to limit its scope.

EXAMPLES Example I Preparation of Extracts

Parenchyma Tissue Extract Preparation (PTE) from Spinach Leaves (Spinacia oleraceae)

The first step of the process involves the homogenization by mechanical pulverization. Leaves and needles of spinach were homogenized by mechanical pulverization at 4° C. The homogenization can be performed between 0° C. and 35° C., but most preferably under 4° C. to avoid any degradation of the tissue during the procedures. The tissue were homogenized in a homogenization buffer composed of 50 mM phosphate buffer pH 7.5 and 10 mM sodium chloride. Taking spinach as a reference plant, the wet weight ratio of plant leaf tissues (g)/volume of buffer (ml) was of about 1/2. When the plant source varies, the buffer volume varies accordingly as known in the art, based among other things, on the water content of the plant. The plant was mixed with the buffer and homogenized in a commercial blender for about 2 minutes in order to obtain PTE.

Mesophyll Cell Extract (MCE) Preparation

Cell debris and soluble components from PTE homogenates were separated by continuous centrifugation at about 3000×g. Application of an equivalent mechanical pressure can replace the centrifugation. The centrifugation system allowed for the filtration of the homogenate based on the size of its cell debris, as the centrifugation means was provided with a 60 μm filter, therefore producing a filtrate (MCE). As a general guideline, the filter to be used with the filtration can be of 40 μm to 200 μm, preferably from 50 μm to 100 μm, and more preferably from 50 μm to 80 μm.

Example II Reduction of the Harmful Effect of Smoke on the Immune Response

Introduction of Buffer and Plant Extracts into the Cigarette Filter

PTE and MCE were introduced directly into the cigarette filter with a 3 ml syringe, 5 mm deep in the middle of the open surface of the filter, so as to saturate the filter with 0.8 ml of PTE, MCE or buffer. The treated filters were allowed to dry, by techniques known in the art, for 24 hours at room temperature before analysis.

Cigarette Smoke Extract

Commercial cigarettes with filters (Bailey's™, toxic emission/unit of tar and nicotine of 15-34 mg and 1.4-2.9 mg respectively) were used in this study. To mimic the average smoke puff, an all-glass impinger-30 (AGI-30) (CIE) filled with 20 ml of sterile PBS buffer with an air-flow of 12.5 l/min and a critical orifice of 15 μm was used. This apparatus mimics conditions allowing the smoke to reach the lungs [May Kr et al., 1957, Br. J. Ind. Med.; 14:287-297]. Two cigarettes were smoked (55 ml/puff, 2 s duration/puff in 30 s intervals) with a mean time of 7.5 min per cigarette and a mean number of 15 puffs. The smoke generated is transferred to the AGI-30 to be diffused through the PBS buffer. A concentration of 3% cigarette smoke extract (CSE) was used to treat the cells with the 20 ml of PBS buffer representing 100% inhibition of macrophage activity. A control with ambient air was also performed using the same apparatus.

Cells

The rat alveolar macrophage (AM) cell line NR8383 exhibits similar functions than freshly isolated AM [Heilmke, et al., 1989, In Vitro Cell Dev. Biol.; 25:44-48; Sirois, et al., 2000, J. Immunol.; 164: 2964-2970]. NR8383 cells were maintained in Ham's F-12 media (Invitrogen Canada Inc, Burlington, ON, Canada) as previously described (Selvay, J. W. 1986, Prog. Clin. Biol. Res. 213:521-536). All experiments were performed in RPMI-1640 medium with 5% FBS, 1% Hepes buffer, 1% penicillin-streptomycin (Invitrogen Canada Inc). After 2 h adherence at 37° C. in 96-well plates (Falcon™; Becton Dickinson Labware, Lincoln Park, N.J.). AM were treated with 3% CSE or air control for 20 h and cell-free supernatants were recovered and stored at −70° C. for future analysis of cytokine content.

Cytokine Release

IL-10, TNFα, and MCP-1 levels were measured in cell-free supernatants using ELISA kits for rat (BD Bioscience Pharmingen, Mississauga, ON, Canada) according to the manufacturer's instructions. Plates were read on a THERMO_(max)™ microplate reader (Molecular Devices, Menlo Park, Calif.). Tobacco smoke extract did not interfere with the ELISA assay.

Cytotoxic Assay

AM were treated with tobacco smoke extract for 20 h and washed before performing the cytotoxic assay toward tumour cells. AM cytotoxic activity was measured using a ⁵¹Cr-release assay, as previously described [Bissonnette et al., 1990, J, Immunol. 145:3385-3390], on WEHI-164 cells (fibrosarcoma target cells). WEHI-164 cells were incubated 90 min with 100 μl of ⁵¹CrNaCrO₄ (Amersham Bioscience, Piscataway, N.J.) and washed three times. AM and WEHI-164 were co-cultured at a ratio of 50:1 AM:WEHI-164 in a U-bottom 96-wells for 18 h in 5% CO₂ atmosphere at 37° C. All plates were done in triplicate. Plates were spun (1000 rpm, 5 min) to obtain supernatants, and the radioactivity was measured in cell-free supernatants. ⁵¹Cr total release (TR) was measured by adding 0.01% Triton X-100™ to target cells and ⁵¹Cr spontaneous release (SR) was measured in supernatant of target cells exposed to medium. Percent of cytotoxicity was calculated using the formula:

$\frac{{{cpm}\mspace{14mu} {in}\mspace{14mu} {presence}\mspace{14mu} {of}\mspace{14mu} {AM}} - {SR}}{{TR} - {SR}} \times 100$

Statistical Analysis

Statistical analysis for cytokines release and cytotoxicity data were made using an ANOVA table followed by a Fisher's post-hoc test to determine statistical significance between groups using StatView™ 5.0 (SAS Institute inc., Cary, N.C.). Differences were considered significant when p<0.05.

Inhibition of AM Mediator Release by Tobacco Smoke and Modulation by Plant Extracts

Concentrations of CSE ranging from 0.1 to 10% were evaluated for their toxicity on a 20 h exposure period. Three percent CSE was identified as the percentage to be used since it inhibited AM cytokine release without altering cell viability, as measured by trypan blue exclusion assay.

To investigate the modulatory effect of plant extract on tobacco smoke-induced AM inhibition, AM were exposed to 3% CSE for 20 h, in the presence of different concentrations of PTE and MCE. The production of different mediators, pro-inflammatory (TNFα), anti-inflammatory (IL-10), and chemokine (MCP-1), was measured in cell-free supernatants. CSE alone significantly inhibited TNFα production by 43.4±5.6% (FIG. 1 a). The addition of low concentrations (0.0025%) of PTE and MCE to CSE reversed this inhibition. Interestingly, the presence of high concentrations (0.025%) of PTE and MCE in CSE stimulated the release of TNFα by AM to levels above those of non-treated AM. The addition of 0.025% PTE and MCE to AM without CSE (data not shown) also stimulated TNFα release (2255±907 and 1973±542 pg/ml, respectively).

CSE reduced by 67.0±11.7% IL-10 production by AM. This CSE-induced reduction was also inhibited by the presence of plant extracts during the treatment (FIG. 1 b). High concentration (0.025%) of MCE in the presence of CSE significantly increased IL-10 production (control, 10.9±2.6 pg/ml, compared with MCE, 20.1±7.1 pg/ml), but was not significantly different from the treatment with MCE alone (26.8±8.3 pg/ml, data not shown). Similar data were observed with MCP-1 production (FIG. 1 c), although the inhibition caused by CSE was not as strong (21.2±2.8%) of the one encountered with IL-10. The addition of 0.025% MCE without CSE significantly increased MCP-1 production by AM (17.4±0.9 ng/ml, data not shown). Our data clearly show that plant extracts (PTE and MCE) can eliminate the inhibitory effects of tobacco smoke on AM inflammatory mediators production.

Inhibition of AM Cytotoxic Activity by Tobacco Smoke and Modulation by Plant Extracts

One of the major roles of AM is the eradication of tumoral cells by exerting a cytotoxic effect on those cells. Thus, in order to determine the effect of tobacco smoke on AM cytotoxic activity, AM cells were cultured as described previously and treated with CSE for 20 h before washing for cytotoxic assays. Three percent CSE significantly inhibited AM cytotoxic activity towards tumoral cells (FIG. 2). The addition of PTE to CSE did not modulate CSE-induced inhibition of AM cytotoxic activity (FIG. 2), indicating that PTE has no direct effect on AM cytotoxic function under these conditions. However, the addition of MCE to CSE abrogated its inhibition of AM cytotoxic activity. This shows that MCE can directly stimulate AM cytotoxic activity. Indeed, treatment of AM with MCE (0.025%) without CSE significantly increased AM cytotoxic activity towards tumoral cells by 39.6±10.5% (data not shown).

Modulation of AM Mediator Production by Plant Extract-Containing Cigarette

The results presented above showed the reversibility of tobacco smoke inhibition of AM inflammatory and cytotoxic activities when AM are exposed to plant extracts. However, the plant extract itself also stimulated AM cytokine release. In order to further investigate the mechanism involved, PTE and MCE were introduced in cigarette filters, thus avoiding direct contact between AM and the plant extract.

To determine the effect of plant extracts-containing cigarette filters on the noxious effects of cigarette smoke, AM were treated for 20 h with 3% CSE from cigarettes having a plant extract-containing filter. The production of TNFα, IL-10, and MCP-1 was measured in cell-free supernatants as previously described. CSE from cigarettes having a buffer-treated filter significantly inhibited the production of all three mediators by AM (FIG. 3), including a decrease of 43.3±7.7% in TNFα production. The presence of high concentrations (0.25%) of PTE and MCE in cigarette filter abrogated this inhibitory effect of cigarette smoke, whereas no effect was noted by low concentrations (0.025%) (FIG. 3 a). In contrast, the inhibition of IL-10 production by cigarette smoke was eliminated in cigarettes having a plant extract-containing filter, regardless of the concentration and the type of plant extract (PTE or MCE) used (FIG. 3 b). Cigarette smoke also significantly reduced MCP-1 production by AM, but only high concentrations (0.25%) of MCE in cigarette filters abrogated this inhibition (FIG. 3 c). Hence, the production of TNFα, IL-10 and MCP-1 by AM was not significantly altered by tobacco smoke from cigarettes having a filter treated with 0.25% MCE when compared with air control.

Modulation of AM Cytotoxic Activity by Plant Extract-Containing Cigarette

AM were treated with CSE from plant extract-containing cigarettes for 20 h and their capacity to kill tumoral cells (fibrosarcoma cells) was investigated. Treatment of AM with CSE from cigarettes having a buffer-treated filter significantly reduced (14.5±5.9%) AM cytotoxic activity (FIG. 4), with similar levels than CSE from cigarettes having an untreated filter (FIG. 2). The presence of high concentrations (0.25%) of PTE in the cigarette filter abrogated the inhibitory effect of the CSE on AM cytotoxic activity, whereas small concentrations (0.025%) did not have a significant effect. In contrast, the presence of both low and high concentrations of MCE in the cigarette filter eliminated the inhibitory effect of CSE on AM cytotoxic activity (FIG. 4). Thus, the presence of plant extract in the filter of cigarettes eliminated the inhibition of AM cytotoxic activity towards tumoral cells caused by tobacco smoke.

Table 1 summarizes the significant effects of the presence of plant extracts in cigarette filters on the production of inflammatory mediators by AM, and on the cytotoxic activity of AM towards tumoral cells. Low concentration of PTE and MCE abrogated tobacco smoke inhibition of AM cytokine production, whereas high concentration of MCE stimulated the release of TNFα, IL-10, and MCP-1. Given that PTE and MCE stimulated AM cytokine release, the elimination of tobacco smoke inhibition can be due, at least in part, to the stimulation of cytokine release by PTE and MCE.

TABLE 1 Data summary of the effects of plant extracts on tobacco smoke-induced inhibition of AM functions. TNFα IL-10 MCP-1 Cytotoxicity Air control — — — — CSE + buffer ↓ ↓ ↓ ↓ CSE + PTE 0.0025% — — — ↓ CSE + PTE 0.025% ↑ — — ↓ CSE + MCE 0.0025% — — — — CSE + MCE 0.025% ↑ ↑ ↑ — AM were treated with cigarette smoke extract (CSE) in the presence or not of plant extract (PTE and MCE) for 20 h and cytokine release was measured in cell-free supernatants, or cells were washed and the cytotoxic activity was investigated. The overall effect on the production of cytokines and cytotoxicity as compared to air control is expressed as follow: inhibition: ↓; increase: ↑; no effect: —.

Given the filtrating capacity of plant extracts and their interaction with the substances present in cigarette smoke, the effects of the presence of PTE and MCE in cigarette filters were evaluated with regards to the toxic and cytotoxic effects of tobacco smoke. A concentration of 0.25% MCE in cigarette filter eliminated the inhibitory effect of tobacco smoke on mediator release by AM and the cytotoxic activity of AM towards tumoral cells (Table 2). PTE (both at 0.25% and 0.025%) and 0.025% MCE were less effective but still presented a significant effect. PTE had a better filtrating capacity than MCE. This suggests that the removal of a larger concentration of total tobacco smoke components is not as effective as the removal of specific components, such as performed by MCE.

TABLE 2 Data summary of plant extract treated cigarette filters on AM functions. TNFα IL-10 MCP-1 Cytotoxicity Air control — — — — CSE, buffer filter ↓ ↓ ↓ ↓ CSE, PTE 0.025% in filter ↓ — ↓ ↓ CSE, PTE 0.25% in filter — — ↓ — CSE, MCE 0.025% in ↓ — ↓ — filter CSE, MCE 0.25% in filter — — — — AM were treated with cigarette smoke extract (CSE) from cigarettes with filter treated with plant extract (PTE or MCE) or buffer for 20 h and cytokine release was measured in cell-free supernatants, or cells were washed and the cytotoxic activity was investigated. The overall effect on the production of cytokines and cytotoxicity as compared to air control is expressed as follow: inhibition: ↓; no effect: —.

To our knowledge, this is the first study demonstrating the reduction of the harmful effects of cigarette smoke on the immune response by plant extracts.

Example III Effect of Plant Extract on the Concentration of Heavy Metals in Cigarette Smoke

Concentration of heavy metals cadmium, lead, nickel, chromium and mercury was determined in the smoke of regular and king size cigarettes equipped with regular filters and with plant extract-containing filters. FIG. 5 shows that there is significant reduction in the concentration of heavy metals concentration in smoke from cigarettes equipped with a plant extract-containing filter when compared with the smoke from cigarettes equipped with a regular filter.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims. 

1-39. (canceled)
 40. A filter for reducing the toxicity or cytotoxicity of a fume, wherein the filter comprising a carrier material and a plant extract comprising at least one of a chlorenchyma tissue extract and a mesophyll cell extract.
 41. The filter of claim 40 wherein the plant extract is selected from the group consisting of: an algae extract, a bryophyte extract, and vascular plant extract.
 42. The filter of claim 41 wherein the vascular plant extract is selected from the group consisting of: a lettuce extract and a spinach extract.
 43. The filter of claim 40 wherein the plant extract is a Spinacia oleraceae extract.
 44. The filter of claim 40 wherein the plant extract comprises intact membrane constituents.
 45. The filter of claim 40 wherein the fume is selected from the group comprising a combustion gas, an environmental gas, an automotive gas, a domestic gas, an industrial gas, and smoke.
 46. The filter of claim 45 wherein the gas is tobacco smoke.
 47. The filter of claim 40 wherein the carrier material is a porous material.
 48. The filter of claim 40 wherein the composition further comprises at least one of an activated charcoal and an ion exchange resin.
 49. The filter of claim 40 wherein the carrier material comprises a cellulose.
 50. The filter of claim 40 wherein the plant extract has a total chlorophyll concentration of between 0.03 and 1100 ng/g of dried plant extract.
 51. The filter of claim 40, wherein the plant extract has a total chlorophyll concentration of between 0.05 and 50 ng/g of dried plant extract.
 52. The filter of claim 40, wherein the concentration of the plant extract in the carrier material is between 0.005 and 200 mg/cm³ of carrier material.
 53. (canceled)
 54. The filter of claim 40 being adapted to fit a cigarette. 55-58. (canceled)
 59. A cigarette comprising the filter of claim
 40. 60-61. (canceled) 